Serum starvation improves transient transfection efficiency in differentiating embryonic stem cells

Control of genetic expression is a critical issue in the field of stem cell biology, where determining a cell fate or reprogramming adult somatic cells into pluripotent cells has become a common experimental practice. In turn, for these cells to have therapeutic clinical potential, techniques for controlling gene expression are needed that minimizes or eliminates the risk of oncogenesis and mutagenesis. Possible routes for achieving this outcome could come in the form of a transient nonviral gene delivery system. In this study, we improved the efficiency of transient gene delivery to differentiating murine embryonic stem (ES) cells via serum starvation for 3 days before transfection. The transient expression of a constitutively‐controlled plasmid increased from ∼50% (replated control) to ∼83% when transfected after 3 days of serum starvation but decreased to ∼28% when transfected after 3 days in normal high serum‐containing media. When probed with a liver‐specific reporter, Cyp7A1, expression increased from ∼1.4% (replated control) to ∼3.7% when transfected after 3 days of serum starvation but decreased to ∼0.7% when transfected after 3 days in high serum‐containing media. Cy3‐tagged oligonucleotides were used to rapidly quantify DNA uptake and predict ultimate transfection efficiency. This study suggests that modifications in media serum levels before transfection can have a profound effect on improving nonviral gene delivery. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010